Photoaffinity labeling and binding studies reveal the existence of two types of phencyclidine receptors in the NCB-20 cell line

Abstract

The mouse neuroblastoma-Chinese hamster brain hybrid cell line NCB-20 is the only clonal cell line in which binding studies indicate the presence of phencyclidine (PCP) receptor-like sites. We report here that polypeptide components of NCB-20 cell PCP sites were identified with the photolabile PCP derivative [ 3 H]N-[1-(3- azidophenyl)cyclohexyl]piperidine ([ 3H]AZ-PCP). The pharmacological selectivity of [ 3H]AZ-PCP binding (under reversible conditions) was similar to that observed for [ 3 H]N-[1-(2- thienyl)cyclohexyl]-piperidine ([ 3H]TCP) binding to NCB-20 cell membranes. Inhibition of [ 3H]TCP binding by AZ-PCP, dexoxadrol or MK-801 was biphasic, suggesting the presence of two types of PCP sites on NCB-20 cells. Photolysis of NCB-20 cell membranes pre-equilibrated with [ 3H]AZ-PCP, followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), revealed the presence of 5 major labeled bands ( M r 90,000, 68,000, 49,000, 40,000 and 33,000), a pattern similar to that observed for rat brain membranes. MK-801 and d-2-amino-5-phosphonovaleric acid ( d-(−)-AP5) selectively inhibited the labeling of M r 68,000 and 90,000 polypeptides. These results indicate that the labeled bands represent constituents of at least two different PCP binding proteins. The M r 68,000 and 90,000 components appear to correspond to a high-affinity site, which comprises approximately 20% of total [ 3H]TCP sites in these cells, and exhibits the pharmacology expected for the PCP receptor of the N- methyl- d-aspartate (NMDA)-gated channel.